Sweeteners are widely used both by consumers and by the food and beverage industry. Consumers use them as an ingredient in various food items and also as a means for customizing the sweetness of beverages, fruit, yogurt, and the like. The food and beverage industry uses such sweeteners in prepared beverages and other food items. Such sweeteners include both caloric and low-caloric sweeteners. Caloric sweeteners include sucrose, fructose, and glucose. Recently, low-calorie (or non-calorie) sweeteners have gained increased popularity. In many instances, they can be used as substitutes for caloric sweeteners and are often referred to as “sugar substitutes”. Common sugar substitutes include saccharin, aspartame, and sucralose. In parts of Asia, compounds extracted from stevia rebaudiana (hereinafter stevia) plants have also been used as sugar substitutes for some time. For example, Japan has marketed stevia as an alternative to artificial sweeteners since 1970. In the 1980s, China began producing stevia commercially, becoming the main supplier to Japan.
There is an increasing interest for natural sweeteners. This interest stems partially from increasing consumer demand for such products, but also from the rise of a variety of businesses selling natural products and requiring suppliers of such products to certify that natural ingredients are used in any products being supplied.
Numerous extraction methods to produce stevia extracts rich in steviosides and rebaudioside A have been described. Most commercial processes used by the stevia industry consist of: water, solvent, and/or supercritical gas extraction; addition of multiple solvents and chemicals such as HCl, NaOH, CaCO2, methanol, H3PO4, NaCl; decoloration (fading); purification using ion-exchange resins; electrolytic techniques; or precipitating agents involving numerous complex steps.
In general, the methods of preparing stevia extracts employed by the stevia industry are complex and involve numerous steps, which can be quite costly and time consuming. In addition, these methods have often been directed towards the separation of individual glycosides such as steviosides, rebaudioside-A, or mixtures thereof, and generally involve the use of organic chemical solvents (e.g., methanol and butanol) and synthetic adsorbents for extraction steps, selective recrystallization and purification. The use of such chemicals are not recommended by the global food industry and are not considered green or environmentally friendly. Moreover, even though the use of ethanol as an extraction solvent is permitted by the food industry, it has been proposed that ethanol extraction may change the taste profile of stevia extracts. Therefore, it is recommended that the stevia industry use non-ethanol extraction processes to avoid having taste issues.
Most of the research and development in the stevia industry is focused on developing extraction methods that yield a product with the highest possible sweetness and lowest possible bitterness. Of the various compounds present in stevia extracts, rebaudioside A is generally considered to exhibit the greatest utility as a sugar substitute. Thus, most of the stevia industry has developed complex methods involving sophisticated equipment in order to produce concentrations of rebaudioside A at 95% or higher, or to reach concentrations of at least 90%, but preferably 95%, for both stevioside and rebaudioside A combined.
Although stevia leaves have been predominantly studied for their sweetening properties, they are composed of, by dry weight: about 6.2% protein; about 5.6% lipids; about 52.8% total carbohydrates; about 15% steviosides and about 42% of water-soluble substances (Kinghorn et al., 1992). Additionally, stevia leaves are a significant source of potentially health benefiting phytochemicals. Over 100 stevia-derived phytochemicals have been discovered thus far. The constituents of stevia leaves can be divided into two categories: sweetening constituents and non-sweetening constituents.
The sweetening constituents represent about 14% by weight of dried leaves and are mostly diterpene glycosides based on the kaurene skeleton. In particular, steviol glycosides are responsible for the sweetening properties of stevia. The most abundant form of steviol glycosides in stevia leaves are steviosides followed next by rebaudiosides. The rest of the sweetening compounds are present in much smaller quantities. Stevioside was isolated in 1931 and has also been produced from the hydrolysis of stevioside as well as isosteviol (Bridel et al., 1931). Subsequent studies have led to the isolation of seven other sweet glycosides of steviol. Among these, rebaudioside A is considered the sweetest and the most stable, and it is less bitter than stevioside. Rebaudioside A represents a much smaller percentage of the total weight of stevia leaves and thus chemical processes aimed at producing higher yield of rebaudioside A (Reb A) have been developed (Dobberstein et al., 1986). Rebaudioside E (Reb E) is as sweet as stevioside, and rebaudioside D (Reb D) is as sweet as rebaudioside A, while the other glycosides are less sweet than stevioside (Crammer and Ikan, 1986; Crammer and Ikan, 1987). The sweetness of high potency sweeteners compared to sucrose are summarized in Table 1 (Crammer and Ikan, 1986).
TABLE 1Sweetness of high potency sweeteners inSucrose Equivalence (% SE) in waterSweetener% SEAspartame16.0Acesulfame-K11.6Sucralose13.0Neotame15.1Cyclamate-Na15.2Saccharin-Na10.1JSSG10.1Rebaudioside D10.1Neohesperidin Didydrochalcone9.8Monoammonium Glycyrhisinate7.3
The sweetening potency of the different glycosides from stevia compared to sucrose. Stevioside is the most prevalent glycoside in stevia, comprising 6-18% of stevia leaves. Typical proportions, on a dry weight basis, for the four major glycosides found in the leaves of wild stevia plants are: 0.4-0.7% dulcoside A; 1-2% rebaudioside C; 2-4% rebaudioside A; and 5-10% stevioside (Wood et al., 1955). Table 2 summarizes the sweetness of the principal steviol glycosides found in stevia leaves compared to sucrose.
TABLE 2Comparison of the sweetness potential of the principal steviolglycosides found in stevia leaves (compared to sucrose).Sweetness comparedSteviol glycosideto sucroseDulcoside A50-120 foldRebaudioside A250-450 foldRebaudioside B300-350 foldRebaudioside C50-120 foldRebaudioside D240-450 foldRebaudioside E150-300 foldSteviobioside100-125 foldStevioside300 fold
The non-sweetening constituents of stevia leaves that have been identified include labdanediterpene, triterpenes, sterols, flavonoids, volatile oil constituents, pigments, gums and inorganic matter (Kinghorn et al., 1992). Table 3 summarizes the main non-sweetening constituents found in stevia leaves (Bridel et al., 1931).
TABLE 3Stevia-derived molecules classified by chemical group and affinityGlycosylated diterpeneSteviol (ent-13-hydroxy kaur-16-en-19-oic acid)derivativesSteviosideSteviolbiosideRebaudioside ARebaudioside BRebaudioside C (Dulcoside B)Rebaudioside DRebaudioside EDulcoside AEssential oilsβ-caryophylleneTrans β-tarneseneα-humuleneδ-cadienecaryophyllene oxideNerolidolLinalolα-terpineolTerpinen-4-olSterol derivativesStigmasterolβ-sitosterolCampesterolFlavonoidsGlucosyl-4′-O-apigeninGlucosyl-7-O-luteolinRhamnosyl-3-O-kaempferolQuercetinGlucosyl-3-O-quercetinArabinosyl-3-O-quercetin5,7,3′-methoxyflavone3,6,4′-methoxyflavone
Methods for preparing stevia extracts are known in the art. In general, they involve the following steps: (1) extraction; (2) pre-treatment; (3) separation, and (4) refining. In some cases, stevia leaves are treated with non-polar solvents such as chloroform or hexane to remove essential oils, lipids, chlorophyll, and other non-polar substances. Methods for isolating sweetening compounds from dried stevia leaves usually involve a solid/liquid extraction step followed by a liquid/liquid purifying step. When this method is used, the glycosidic extract has a pronounced aftertaste which can be attributed to the presence of oils, tannins, and flavonoids (Phillips, 1987).
The extraction processes generally involve an initial liquid extraction using a solvent such as water, methanol, or a mixture of the two. Pressurized fluid extractions using water or methanol have been studied for the extraction of stevioside from stevia leaves. The results of these studies indicated that methanol has a better extraction ability than water within at temperatures between 110-180° C. (Pol et al., 2007). Supercritical fluid extractions with CO2 and a co-solvent (e.g., methanol, ethanol, and acetone) have been suggested (Tan et al., 1988; Pasquel et al., 2000). It was claimed that the quality of the glycosidic fraction obtained thereby, in terms of its sweetening capacity, is higher in comparison with the one obtained with conventional methods in the art. Other groups have proposed using more complex microwave-assisted extraction (MAE) methods for isolating stevioside and rebaudioside A (Jaitak et al., 2009).
Besides the choice of solvent and the mode of extraction used, other parameters are known to affect the quality of the stevia extraction as well as the quantity of impurities (e.g., protein, pigments, pectin, and flavonoids) present therein. These parameters include pH and temperature. For example, it is known that the yield of sweetening compounds is usually higher when using higher extraction temperatures; however, this is known to lead to an undesirable crude extract which is of lower quality (containing a higher concentration of impurities such as undesirable bitter high molecular weight compounds). To counter this problem, the level of impurities can be reduced by precipitation with CaCO3 at pH of 10. The use of ultrasonic waves during extraction has also been proposed to increase recovery (Shoji et al., 1999). Higher extraction temperatures can also lead to an increase in pigment impurity concentration. For example, a column-based extraction performed at 50° C. can lead to nearly double the concentration of pigment impurities in comparison with a column-based extraction performed at 25° C. Furthermore, an extraction temperature of 4° C. can reduce the quantity of impurities to about 70% of that of a corresponding extraction performed at 25° C. Thus, according to the teachings of the prior art, the level of pigments (and other undesirable such as bitter high molecular weight compounds) is higher when extracted at high temperatures (e.g., 25° C. to 50° C.) than at lower temperatures (e.g., 4° C.). Consequently, the prior art teaches that a stevia extract is of better quality when extracted at low temperatures.
While water-based extraction methods for stevia have been previously described, these methods tend to be rather complicated, less or not efficient, costly and require the use of organic chemical solvents. A water-based stevia extraction method that qualifies as “organic certifiable” and which does not require the addition of other solvents or chemicals, has yet to be developed.
A variety of pre-treatment strategies have also been employed in the stevia industry to enhance the efficiency of the extraction process. However, these additional strategies necessitate an additional step to the extraction process, and therefore can increase the cost and time of the extraction and/or require the use of complex apparatuses. For example, the use of inorganic salts such as Ca(OH)2 is commonly used. Other inorganic salts that are sometimes used include sulfates of potassium, aluminum, and iron (Fuh et al., 1990; Adduci et al., 1987; Yokoyama and Sugiyama, 1990). Aluminum salts have been used to remove pigment from the crude extract. Heat evaporation processes have been also used. Ceramic microfiltration provides chemical-free pre-treatment to remove large suspended impurities from the crude extract. Membrane separation technology can substitute a heat-evaporation process. Ultrafiltration is the most common method for separation of remaining larger molecular weight impurities from the clarified extract (Kutowy et al., 1998; Fuh et al., 1990. Liu et al., 1991).
Purification steps involving ion exchange and adsorption column chromatography have been used for isolating sweeteners. However once again, these techniques are often complex (e.g., requiring numerous steps), time-consuming, costly, directed to the separation of individual glycosides, and/or often involve the use of organic chemical solvents and/or other synthetic chemicals which are not recommended by the food industry. Briefly, the use of strongly acidic cation-exchange resin, strongly basic exchange resin, as well as mixed bed columns of weakly acidic cation-exchange resin and weakly basic anion-exchange resin have been used in complex processes to isolate stevioside with a purity of 80-87% and a recovery of about 79% (Cheng and Chang, 1985). Furthermore, a bifunctional polymeric adsorbent was stated to achieve higher recovery for rebaudioside A than the commercial resin with stable and easier regeneration capacity (Shi et al., 2002). In other studies, active carbon has also been used for the adsorption of stevioside, in which an optimal ethanol concentration for stevioside desorption was reported to be 60-65%. A significantly higher amount of rebaudioside A was recovered with active carbon from an aqueous solution (Chang et al., 1980). Nanofiltration has been used to refine and concentrate the permeate following an ultrafiltration step. In this regard, a higher temperature nanofiltration was shown to remove certain low molecular weight compounds that might contribute to the bitter taste of the final product (Kutowy et al., 1998). Flavonoids such as apigenin-4′-o-glucoside, quercitrin, and others were shown to be present in water-based stevia extracts (Rajbhandari et al., 1979). These flavanoids are yellowish in color, have a bitter taste, and are of lower molecular weight than stevia glycosides, allowing them to permeate through the nanofiltration membranes. Subsequent purification steps are generally employed to improve yield and/or purity involving an additional extraction in polar organic chemical solvent, decoloration (removal of colored pigments), coagulation, ion exchange chromatography and crystallization (Pasquel et al., 2000; Kinghorn et al., 1985). The filtrate can then be re-precipitated and re-filtered to yield a 90% pure stevioside extract (Adduci et al., 1987; Yokoyama et al., 1990). Thus, many complex and/or costly techniques have been previously employed or are still used in the purification of stevia. 
Commercially available stevia extracts contain a high percentage of the glycoside diterpenes stevioside (CAS no. 57817-89-7) and rebaudioside A (CAS no. 58543-16-1), the principal sweetening compounds, and smaller amounts of other steviol glycosides. The exact composition of the extracts depends on the composition of the stevia leaves from which they originate, which in turn are influenced by factors such as soil, climate, cultivation methods, harvest time, as well as on manufacturing process including the extraction and purification methods used. The impurities present in stevia extracts are primarily due to other compounds that are co-extracted from stevia leaves, such as pigments and saccharides. Regulatory submissions from countries in different parts of the world suggest that the main components of commercially available extracts of stevia contain, as the main components, stevioside and rebaudioside A. The amounts of these compounds range from about 10-70% for stevioside and about 20-70% for rebaudioside A. Furthermore, most commercially available stevia extracts have a total steviol glycoside content of more than 90%, with the two main steviol glycosides (stevioside and rebaudioside A) making up about 80% of the extracts (Wallin, 2004).
According to Phillips et al., (1987) the bitter aftertaste in stevia extracts is due to the presence of essential oils, tannins, and flavonoids. However. Soejarto et al., (1983) concluded that the sesquiterpene lactones are responsible for the bitter aftertaste, while Tsanava et al., (1991) suggested that caryophyllene and spathulenol contribute decisively to the aftertaste. Nevertheless, along with providing sweetness, it appears that at least a portion of the aftertaste is attributable to stevioside and rebaudioside A, although the contribution of rebaudioside A is significantly less than that of stevioside (Jakinovich et al., 1990).
Different methods exist for improving the taste of stevia extracts (e.g., diminishing its bitter aftertaste). The methods involve enzymatic modification of stevioside by pullanase, isomaltase (Lobov et al., 1991), β-galactosidase (Kitahata et al., 1989), or dextrin saccharase (Ghanta et al., 2007). Another method involves adding thaumatin, a natural protein that is a low calorie flavor modifier, which is extracted from the fruits of the katemfe (thaumatococcus daniellii) tree from the West African rain forest. Yet another method suggests enriching the extract with rebaudioside A and rebaudioside D. Furthermore, exposure to low pH (acidic conditions including citric acetic and malic tartaric acid) have been reported to enhance the sweetness of stevia extracts. Finally, the addition of plant (Ogawa) extracts which are natural stevia optimizers, have the ability to mask the unpleasant aftertaste of stevia. 
Thus, there remains a need for a method for preparing an organic certifiable stevia extract with high sweetness which is less complex, less costly, less expensive, less time consuming, environmentally friendly and does not employ organic chemical solvents which may be unfit for human consumption, and would otherwise qualify for “organic certification” yet is amenable to large-scale production. The present invention seeks to address these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.